Synthesis of a neurostimulative piperazine

ABSTRACT

The invention describes an improved synthesis for piperazine derivatized with nicotinic acid and a benzyl moiety. The product compounds are useful for treatment of neurological conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/392,489, having an international filing date of 24 Aug. 2010, whichis the national phase of PCT application PCT/US2010/046537 having aninternational filing date of 24 Aug. 2010, which claims benefit of U.S.patent application No. 61/236,477 filed 24 Aug. 2009. The contents ofthe above patent applications are incorporated by reference herein intheir entirety.

TECHNICAL FIELD

The present invention relates to a synthesis method for compounds andtheir salts useful as neurogenesis agents. More specifically, theinvention is directed to a method to prepare disubstituted piperazinescoupled with benzyl and nicotinic acid moieties.

BACKGROUND ART

U.S. Pat. No. 7,560,553, incorporated herein by reference, describesvarious compounds, including the class of compounds whose synthesis isdescribed herein, as neurogenesis agents. Thus, the compounds preparedaccording to the invention method are useful in the treatment of variousconditions which benefit from promoting neurogenesis byproliferation/differentiation of human hippocampal multipotentstem/progenitor cells and neuronal progenitors. Such conditions includeAlzheimer's disease, mild cognitive impairment, dementia, stroke,traumatic brain injury, spinal cord injury, schizophrenia and the like.The synthesis method provided by the invention avoids the use ofcontrolled substances such as benzyl piperazine.

DISCLOSURE OF THE INVENTION

The invention method employs piperazine either protected at one of thering nitrogens or by selective reaction on only one ring nitrogen, and asubstituted nicotinic acid as starting materials and culminates inproviding disubstituted piperazine comprising a benzyl substitution atone of the ring nitrogens. The synthesis may further include conversionof this disubstituted piperazine to a suitable salt. Thus, in oneaspect, the invention is directed to a method to synthesize a compoundof the formula:

wherein

R¹ is alkyl;

R² is H or alkyl;

each R³ and R⁴ is independently alkyl, alkenyl, halo, aryl, heteroaryl,arylalkyl, heteroarylalkyl, NR₂, SR, or OR where R is alkyl or aryl;

n is 0, 1 or 2;

m is 0, 1, 2 or 3;

which method comprises reacting a compound of the formula

where R³, R⁴, m and n are as defined in formula (1) and L is a leavinggroup,

with a compound of the formula

wherein R¹ and R² are as defined in formula (1).

The compound of formula (2) may be prepared by reacting a compound offormula

wherein R³ and n are as defined in formula (1) and L is a leaving group,

with a compound of the formula

wherein R⁴ and m are as defined in formula (1), and L′ is a leavinggroup, or with a compound of the formula

to form an imine followed by reducing said imine

In turn, the compound of formula (3) may be obtained by reacting acompound of the formula

wherein R³ and n are as defined in formula (1) and L is a leaving group,

with a compound of the formula

wherein Pr is a protecting group,

followed by removing the protecting group or selectively coupling withone nitrogen using unprotected piperazine. The reaction employing aprotecting group may be done either by contracting the compound offormula (4) with the protected piperazine in the presence of a peptidecoupling agent or by converting the compound of formula (4) to thecorresponding benzoyl halide and adding the protected piperazine in thepresence of mild base.

The compound of formula (1) may also be converted to a suitable acidaddition salt such as the sulfate, phosphate, hydrohalide, citrate,fumarate, tosylate, or besylate salt. Both mono and bis salts may beformed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an Optimal Process for Step 1B of Scheme 1.

FIG. 2 shows an Optimal Process for Step 2 of Scheme 1.

FIG. 3 shows an Optimal Process for Steps 3 and 4 of Scheme 1.

FIG. 4 shows an Optimal Process for Step 5 of Scheme 1.

MODES OF CARRYING OUT THE INVENTION

The compound of formula (1) and its salts, and in particular thecompound of formula 1E and its salts have been demonstrated to haveneurogenesis activity as described in the above-referenced U.S. Pat. No.7,560,553. The present invention is directed to an improved method forsynthesis of these compounds as illustrated below in Examples 1-5.

More generally, the synthesis of these compounds comprises the followingsteps.

As shown in Scheme 1, optionally substituted nicotinic acid containing aleaving group at position 2 is reacted with semi-protected piperazine inthe presence of a peptide coupling agent in the presence of mild baseand an appropriate solvent. Typically, the reaction proceeds at ambientconditions to provide a protected form of the compound of formula (3),which is then deprotected in acid in a hydrophilic solvent at slightlyelevated temperatures. The resulting product of formula (3) is reactedwith optionally substituted benzyl containing a leaving group at themethylene portion in the presence of mild base and suitable solvent,also at elevated temperatures to provide the compound of formula (2)which need not be isolated, but reacted with a primary or secondaryamine at elevated temperatures and an appropriate solvent to obtain thecompound of formula (1). The compound of formula (1) may then be reactedwith 1 or 2 mol of acid to obtain an acid addition salt. If step 3 isconducted by substituting a benzaldehyde for benzyl-L′, an imine isformed which is then reduced to the amine using sodium borohydride,sodium cyanoborohydride, sodium triacetoxyborohydride or lithiumborohydride in almost any organic solvent.

Typically, the temperature at which step 1A is conducted is between 20°C. and 30° C.; typical bases include triethylamine or other tertiaryamines and an excess of a semi-polar non-protic solvent such as butylacetate or isopropyl acetate. Step 2 is typically conducted between 50°C. and 60° C. using strong acid such as HCl or sulfuric acid in thepresence of an alcohol solvent. Steps 3 and 4 are conducted between 45°C. and 60° C. for step 3 and between 80° C. and 90° C. for step 4. Step3 is conducted using a mild base such as triethylamine and an aproticsolvent such as acetonitrile or DMSO. Step 4 is also conducted in thepresence of an aprotic solvent.

Step 5 is carried out under conditions dependent on the nature of theacid; either one or two equivalents of the acid may be used to obtain asuitable salt.

In an alternative to step 1A, the compound of formula (3 Pr) may beprepared using step 1B avoiding the use of an expensive peptide couplingagent:

Step 1B is conducted between 60° C. and 70° C. in the presence of abase, such as a tertiary amine, in an excess of a semipolar non-proticsolvent such as butyl acetate or isopropyl acetate. Thus, step 1B isconducted under conditions similar to those of step 1A except that thenicotinic acid is converted to the acyl halide in the presence of SOCl₂.

The remainder of the scheme may remain the same although the yield maybe improved by slightly lowering the temperature at which step 3 isconducted.

As noted above, both R¹ and R² may be alkyl and also alkyl substituentsare included among those optionally present on the nicotinic acid andbenzyl moieties; further, NR₂SR OR may be substituents where R is alkyl.The substituents R³ and R⁴ may also be alkenyl.

As used herein, the terms “alkyl,” and “alkenyl” include straight-chain,branched-chain and cyclic monovalent hydrocarbyl radicals, andcombinations of these, which contain only C and H when they areunsubstituted. Examples include methyl, ethyl, isopropyl, isobutyl,cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butenyl, and the like. Thetotal number of carbon atoms in each such group is sometimes described,e.g., when the group can contain up to 10 carbon atoms it can berepresented as 1-10C or as C1-C10 or C1-10. In general, it is preferredthat one of R¹ and R² be H and the other alkyl with a maximum of 10 or 8carbon atoms, and R³ and R⁴ when embodied as alkyl or alkenyl typicallycontain a maximum of 8 or 6 carbon atoms.

Typically, the alkyl and alkenyl substituents of the invention contain1-10C (alkyl) or 2-10C (alkenyl). Preferably they contain 1-8C (alkyl)or 2-8C (alkenyl). Sometimes they contain 1-4C (alkyl) or 2-4C(alkenyl). A single group can include more than one double bond; suchgroups are included within the definition of the term “alkenyl.”

Alkyl and alkenyl groups may be unsubstituted or substituted to theextent that such substitution makes sense chemically from the standpointof the synthesis procedure and the properties of the end product.Unsubstituted forms are preferred.

As further noted above, R³ and R⁴ may also be aryl or heteroaryl.

As used herein, “aryl” refers to a monocyclic or fused bicyclic moietyhaving the characteristics of aromaticity; examples include phenyl andnaphthyl. Similarly, “heteroaryl” refers to such monocyclic or fusedbicyclic ring systems which contain as ring members one or moreheteroatoms selected from O, S and N. The inclusion of a heteroatompermits aromaticity in 5-membered rings as well as 6-membered rings.Typical heteroaromatic systems include monocyclic C5-C6 aromatic groupssuch as pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, thienyl, furanyl,pyrrolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl andimidazolyl and the fused bicyclic moieties formed by fusing one of thesemonocyclic groups with a phenyl ring or with any of the heteroaromaticmonocyclic groups to form a C8-C10 bicyclic group such as indolyl,benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl,benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl,quinoxalinyl, cinnolinyl, and the like. Any monocyclic or fused ringbicyclic system which has the characteristics of aromaticity in terms ofelectron distribution throughout the ring system is included in thisdefinition. It also includes bicyclic groups where at least the ringwhich is directly attached to the remainder of the molecule has thecharacteristics of aromaticity. Typically, the ring systems contain 5-12ring member atoms. Preferably the monocyclic heteroaryls contain 5-6ring members, and the bicyclic heteroaryls contain 8-10 ring members.

Similarly, “arylalkyl” and “heteroarylalkyl” refer to aromatic andheteroaromatic ring systems which are bonded to their attachment pointthrough a linking group such as an alkylene, including saturated orunsaturated, cyclic or acyclic linkers which optionally contain one ormore hetero atoms selected from O and S. Typically the linker is C1-C8alkyl or a C1-C8 heteroalkyl linker. An arylalkyl group may be, forinstance, a phenyl ring and a C1-C4 alkylene where the alkyl orheteroalkyl groups can optionally cyclize to form a ring such ascyclopropane, dioxolane, or oxacyclopentane.

“Alkylene” as used herein refers to a divalent hydrocarbyl group;because it is divalent, it can link two other groups together. Typicallyit refers to —(CH₂)_(n)— where n is 1-8 and preferably n is 1-4, thoughwhere specified, an alkylene can also be substituted by other groups,and can be of other lengths, and the open valences need not be atopposite ends of a chain. Thus —CH(Me)— and —C(Me)₂— may also bereferred to as alkylenes, as can a cyclic group such ascyclopropan-1,1-diyl.

Aryl, heteroaryl, arylalkyl and heteroarylalkyl groups maybeunsubstituted or substituted to the extent that such substitution makessense chemically from the standpoint of the synthesis procedure and theproperties of the end product. Unsubstituted forms are preferred.

“Halo”, as used herein includes fluoro, chloro, bromo and iodo. Chloroand bromo are often preferred.

Suitable leaving groups for L and L′ include halo, such as chloro, IODOor bromo, tosylates (OTs), and triflates OTf). Other suitable leavinggroups include mesylates (OMs), and brosylates (OBr).

Peptide coupling agents includeO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU) as well as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC), N-hydroxybenzotriazole (HOBt), carbonyl diimidazole(CDI), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), N,N′ Dicyclohexyl carbodiimide (DCC), andN-hydroxy succinamide (NHS).

Suitable protecting agents include 9-fluoroenyl methyl carbamate (Fmoc),and t-butyl carbamate (Boc) as well as TBDMS, TMS, TES, TIPS, TBDPS,benzoyl and carbamates or amides in general.

These lists are non-exhaustive and many suitable leaving groups,protecting groups, and peptide coupling agents are known in the art andmany are commercially available.

All these reactions can be done in organic solvents or aqueous organicsolvents such as tetrahydrofuran (THF), dimethyl formamide (DMF),methylene chloride, MTBE, all alkanes, NMP, DMA, EtOAc, and in otherthan for step (2), except for alcoholic solvents).

Preferred embodiments include those wherein R² is H and R¹ is ethyl,propyl, butyl or amyl including the iso forms thereof. Further preferredforms are those wherein m and/or n are 0 or 1, preferably 0. Preferredleaving groups are halo, preferably chloro.

The following examples are offered to illustrate but not to limit theinvention.

Examples 1-5 detail the following series of reactions.

EXAMPLE 1 Preparation of Protected 3E (Step 1A)

A. Chloronicotinic acid (5.0 g) (4E) was charged to a round-bottom flaskfollowed by acetonitrile (anhydrous, 40 mL) and TBTU (1.4 equiv). To theresulting solution was added triethylamine (2.0 equiv) and the mixturewas stirred at ambient temperature for 30 minutes. Boc-piperazine (1.4equiv) was added in portions keeping the temperature inside the flask at<20° C. The reaction was slowly heated to 40° C. and was judged completeby HPLC analysis after four hours.

The reaction mixture was quenched with a saturated NaHCO₃ solution (40mL) and was extracted with isopropyl acetate (IPAc) (2×40 mL). Theorganic layers were combined and washed with a 50% brine solution (40mL). The organic layer was dried over Na₂SO₄, filtered and concentratedto one-fourth of the original volume. The resulting oil turned into athick suspension upon stirring.

Methyl tert butyl ether (MTBE, 100 mL) was added and the resultingsuspension was cooled in an ice-water bath and stirred for one hour. Thesolids were collected by filtration on a Whatman® #1 filter paper andthe filter cake was washed with cold MTBE (20 mL). The solid was driedin a vacuum oven at ambient temperature to afford 6.7 g (65% yield) of3EPr as a light brown solid.

The above reaction was repeated under identical conditions and on thesame scale resulting in 6.5 g (65% yield) of 3EPr.

B. The procedure of paragraph A was repeated using 1 g instead of 5 g ofchloronicotinic acid and corresponding amounts of other reactants, andusing ethyl acetate or isopropyl acetate as solvents. The yields were:

Ethyl acetate: 50% with an HPLC purity of 95.5% (AUC @ 226 nm).

Isopropyl acetate: 80% with an HPLC purity of 97.8% (AUC @ 226 nm).

C. In a modified procedure from paragraph A, chloronicotinic acid (5.0g) was charged to a round-bottom flask followed by IPAc (reagent grade,40 mL) and triethylamine (2.0 equiv). To the resulting solution wasadded TBTU (1.4 equiv) and the mixture was stirred at ambienttemperature for 30 minutes. Boc-piperazine (1.4 equiv) was added inportions keeping the temperature inside the flask at <20° C. Thereaction was stirred at ambient temperature over the weekend and wasjudged complete by HPLC analysis after 50 hours. The reaction mixturewas quenched with saturated NaHCO₃ solution (40 mL) and was extractedwith IPAc (2×40 mL). The organic layers were combined and washed with a50% brine solution (40 mL).

The organic layer was dried over Na₂SO₄, filtered and concentrated toone-quarter of the original volume.

To the resulting oil was added MTBE (100 mL) and the resultingsuspension was stirred at ambient temperature for 5.5 hours and foranother two hours in an ice-water bath. The solids were collected byfiltration on a Whatman® #1 filter paper and the filter cake was washedwith cold MTBE (20 mL). The product was dried in a vacuum oven atambient temperature to afford 6.3 g (61% yield) of 4EPr as a light brownsolid. The HPLC purity was >99.9% (AUC @ 226 nm).

D. The reaction of paragraph C was scaled up to 10 g and went tocompletion after 16 hours. The IPAc extract obtained after aqueousworkup in the same manner as above was divided in two equal parts. Eachpart was reduced to 20 g (≈1:1 IPAc/product by weight) under reducedpressure.

Part 1: To the resulting slurry was added MTBE (100 mL). The suspensionthat resulted was stirred at ambient temperature for 16 hours and foranother two hours in an ice-water bath. The solids were collected byfiltration on a Whatman® #1 filter paper and the filter cake was washedwith cold MTBE (20 mL). The product was dried in a vacuum oven atambient temperature to afford 6.8 g (66% yield) of 4EPr as a light brownsolid. The HPLC purity was >99.9% (AUC @ 226 nm).

Part 2: The process for Part 2 was the same as Part 1 but used heptanesas the antisolvent, resulting in 8.2 g (80% yield) of 4EPr as a lightbrown solid. The HPLC purity was >99.9% (AUC @ 226 nm).

EXAMPLE 2 Preparation of 3EPr by Step 1B

A. Chloronicotinic acid (5.0 g, 31.7 mmol) was charged to a round-bottomflask followed by toluene (anhydrous, 40 mL) and DMF (120 μL, 0.05equiv). The resulting slurry was heated to 55° C. and then thionylchloride (4.6 mL, 2.0 equiv) was added dropwise over five minutes. Theslurry was stirred at 55° C. for three hours during which time gasevolution was observed and the mixture turned homogeneous. A sample wastaken and quenched into methanol containing triethylamine to give themethyl ester for HPLC analysis. Analysis by HPLC showed the conversionto the acid chloride was complete. The flask was fitted for distillationand heated to reflux. Approximately 20 mL of solvent was removed thenthe solution was cooled to ambient temperature. A separate flask wascharged with N-Boc-piperazine (7.1 g, 1.2 equiv), acetonitrile (30 mL, 6vol), and triethylamine (13.3 mL, 3.0 equiv). A slight endotherm wasnoted. The prepared solution of acid chloride was then added at a ratethat maintained the internal temperature below 35° C. The resultingslurry was stirred for one hour at ambient temperature. Analysis by HPLCshowed the reaction to be complete. The reaction mixture was quenchedwith saturated NaHCO₃ solution (20 mL) and the aqueous layer wasextracted with isopropyl acetate (20 mL). The organic layers werecombined and washed with water (10 mL). HPLC analysis of the water washshowed some loss of product to the aqueous layer. The organic layer wasconcentrated to approximately two volumes and then heptanes were added(50 mL) to induce precipitation. The resulting slurry was stirred atambient temperature for 30 minutes, cooled to 0-5° C. for one hour,filtered, and washed with heptanes. The wet cake was then driedovernight under vacuum to give 9.85 g of 3EPr [MDM-W-1(14), 95% yield,99.8 area % by HPLC] as a light yellow solid.

B. The procedure of paragraph A of this Example was conducted using 1.2equivalents of thionyl chloride and 1.1 equivalents of N-Boc-piperazine.The reaction of 2-chloronicotinic acid with thionyl chloride was done at65° C. to better control gas evolution. The reaction of theacid-chloride intermediate and N-Boc-piperazine was done in IPAc insteadof acetonitrile to help prevent precipitation of sodium bicarbonateduring the quench. The reaction yielded 3EPr as an off-white solid[MDM-W-5(8), 9.83 g, 95% yield, >99.9 area % by HPLC].

C. The quench and workup of the reaction with aqueous sodium bicarbonatesolution can lead to an emulsion that requires time to separate.Switching to a water quench alleviated this problem on a small scale;however as the scale increased, a significant rag layer persisted. Therag layer could be dissolved by slightly warming the biphasic mixture to30-35° C.

EXAMPLE 3 Deprotection (Step 2)

A. 1-g of 3EPr prepared in Example 1 or Example 2 was treated with 2equivalents HCl and 5-6N TF 2-propanol at 50° C. The reaction was fondto have gone to completion in 6 hours.

B. The method of paragraph A was repeated with 6.7 g of 3EPr. To asolution of 3EPr (6.65 g) in 2-propanol (5 vol) was added 5-6 N HCl in2-propanol (2 equiv). The reaction was heated to 40° C. and was judgedcomplete by HPLC analysis after four hours. A white suspension formedduring this time.

The reaction was cooled to ambient temperature and the solids werecollected by filtration on a Whatman® #1 filter paper. The filter cakewas washed with 2-propanol (20 mL). The solid was dried under highvacuum to obtain 4.63 g (86% yield) of 3E.HCl as a white solid. The ¹HNMR was consistent with the assigned structure and the HPLC puritywas >99.9% (AUC @ 226 nm).

C. The process of paragraph A was repeated using 11.5 g of 3EPr. To asolution of 3EPr (11.5 g) in IPA (70 mL, 6 vol) was added 5-6 N HCl inIPA (2 equiv). The reaction was heated to 50° C. and was judged completeby HPLC analysis after nine hours. A white suspension formed during thistime.

The reaction was cooled to ambient temperature and the solids werecollected by filtration on a Whatman® #1 filter paper. The filter cakewas washed with IPA (2×15 mL). The solid was dried under high vacuum toobtain 9.01 g (97% yield) of 3E.HCl as a white solid. The ¹H NMR wasconsistent with the assigned structure and the HPLC purity was >99.9%(AUC @ 226 nm).

In each of the foregoing cases, addition of the acid in 2-propanol maybe carried out at higher temperatures, e.g., 55° C. or 60° C. Thisbetter controls the evolution of gas.

D. Compound 3EPr (9.0 g, 27.6 mmol) was charged to a round-bottom flaskfollowed by 2-propanol (5 vol). The slurry was heated to 55° C. duringwhich time the mixture turned homogeneous and 5-6 N HCl in 2-propanol (2equiv) was added dropwise. The reaction mixture was stirred at 55° C.for four hours during which time a thick suspension formed. HPLCanalysis indicated the reaction was complete. The resulting slurry wascooled to ambient temperature and filtered washing with 2-propanol (2vol). The wet cake was dried under vacuum at ambient temperature toprovide 3E [MDM-W-11(3), 6.9 g, 96% yield, >99.9 area % by HPLC].

E. The reaction in paragraph D was scaled six-fold and assessed byreaction calorimetry (RC1, Mettler-Toledo). A gas-flow meter wasconfigured and calibrated to ensure an accurate measurement of gasevolution. Compound 3EPr (56.6 g, 174 mmol) was suspended in 2-propanol(300 mL) and the slurry was heated to 55° C. during which time themixture became homogeneous. Hydrochloric acid (1 equiv) in 2-propanol(3.8 M) was added via an addition pump at a linear rate over 30 minutesduring which time an off-gas was noted and precipitation began. Thereaction was then allowed to stir for 30 minutes before addinghydrochloric acid (1 equiv) at the same rate. The resulting slurry wasstirred for four hours at 55° C. The slurry was cooled to ambienttemperature and filtered washing with 2-propanol to give 44.0 g of alight-yellow solid after drying over the weekend at ambient temperatureunder vacuum [MDM-W-56(1), 97% yield, >99.9 area % by HPLC]. A very mildendothermic thermal profile was observed giving an enthalpy of reactionof −57.8 kJ/mol and an adiabatic temperature change of −9.6 K. The rateof gas evolution was mild. Integration of the mass-flow curve indicated3.9 L of gas evolved during the experiment. The mass-flow curve showedthat the rate of gas evolution decelerated almost immediately upon thediscontinuation of the HCl addition suggesting that gas evolution wasreasonably dose-controlled.

EXAMPLE 4 Conversion to 1E (Steps 3 and 4)

A. A crude sample of 3E prepared in Example 3 was complexed with TFA andreacted with benzaldehyde and the product purified by columnchromatography (2-6% methanol/DCM). Fractions containing the productwere collected and solvent was removed under reduced pressure to obtaincompound 2 G as a thick oil. The ¹H NMR was consistent with the assignedstructure. Since 2E was an oil, 3E was converted to compound 1E in atwo-step telescoped method.

B. To a 0.6 g sample of 3E.HCl in 2-propanol was added triethylamine (2equiv) followed by benzyl chloride (1.2 equiv). The resulting suspensionwas heated to 50° C. when it turned into a clear solution. The reactionwas monitored by HPLC and was judged complete after three hours.

The reaction mixture was cooled to ambient temperature and the solids(TEA.HCl salt) were filtered. To the filtrate was added isoamylamine (10equiv), and the resulting solution was heated to 75° C. The reaction wasmonitored by HPLC and was found to have undergone only 36% conversionafter 48 hours.

C. The procedure of paragraph B was carried out with 3.5 g of 3E.HCl inacetonitrile (20 mL). The reaction was carried out with 1.0 equivalentof benzyl chloride in the presence of triethylamine (3.0 equiv). Thereaction was judged complete by HPLC analysis after stirring at 50° C.for 4.5 hours. The reaction mixture was cooled to ambient temperatureand the solids were filtered. The filtrate was pumped to dryness. Theresidue was dissolved in isoamylamine (20 mL) and was heated to 90° C.The reaction was judged complete by HPLC analysis after 24 hours. Thereaction mixture was cooled to ambient temperature and the solvent wasreduced to adjust the weight of the residue to 9.5 g. To this was addedheptanes (30 mL), resulting in the formation of a light brownsuspension. This was stirred at ambient temperature for one hour and foranother hour in an ice-water bath. The solids were collected byfiltration on a Whatman® #1 filter paper and the filter cake was washedwith cold water (2×20 mL). The product was dried in a vacuum oven,resulting in 3.98 g (70% yield) of compound 1E with a HPLC purityof >99.9% (AUC @ 226 nm).

D. Alternatively, to an 8.0 g sample of 3E.HCl in acetonitrile (48 mL, 6vol) was added triethylamine (2.5 equiv) followed by benzyl chloride(1.05 equiv). The resulting suspension was heated to 50° C. when itturned into a clear solution. The reaction was monitored by HPLC and wasjudged complete after 3.5 hours (3.3% of unreacted 3E.HCl). The reactionmixture was cooled to ambient temperature and the solids (TEA.HCl salt)were filtered.

The filtrate was evaporated to adjust the weight of the solution to 18 g(≠1:1 acetonitrile/product by weight). To this was added isoamylamine(≈4:1 isoamylamine/acetonitrile, 10 equiv of isoamylamine) and theresulting solution was heated to 85° C. The reaction was judged completeby HPLC analysis after 19 hours (3.0% of unreacted 2E). The reactionmixture was cooled to ambient temperature and the solvent was removedunder reduced pressure to adjust weight of the solution to 22 g (1 g ofsolvent per gram of 1E). On cooling a wet solid was obtained and thiswas triturated with heptanes (6 g per gram of 1E). The suspension wasstirred at ambient temperature for 16 hours and the solids werecollected by filtration on a Whatman® #1 filter paper and the filtercake was washed with heptanes (20 mL), followed by water (2×20 mL). Theproduct was dried in a vacuum oven at ambient temperature to afford 7.78g (69% yield over two steps) of 1E as a light brown solid. HPLC puritywas >99.9% (AUC @ 226 nm).

E. Steps 3 and 4 were conducted on a 6 g scale following the procedureset forth above. Compound 3E (6.0 g, 22.9 mmol) was suspended inacetonitrile (30 mL) and triethylamine was added (9.6 mL, 3 equiv)followed by benzyl chloride (2.8 mL, 1.05 equiv). The reaction washeated to 50° C. for 24 hours. HPLC analysis at 20 hours and again at 24hours indicated no further progression (10.4% of 3E remaining) and thereaction was cooled to ambient temperature and filtered to removeammonium salts. The solution was then concentrated under vacuum toapproximately two volumes to give a concentrated solution of crude 2E(80 area % crude purity). Isoamylamine (26 mL, 10 equiv) was then addedand the reaction was heated to reflux (81° C.) for 24 hours. HPLCanalysis at 20 hours and again at 24 hours indicated no furtherprogression (73 area % crude purity) and the reaction cooled to ambienttemperature and was concentrated under reduced pressure to approximately4 volumes. Heptane (35 mL) was then added and the resulting slurrystirred over the weekend. The thin slurry was filtered and washed withwater at which point the solids dissolved leaving nothing on the filterfunnel. The biphasic filtrate was extracted with IPAc and thenconcentrated to an oil. The oil was dissolved in IPA (30 mL); water (36mL) was slowly added until the solution turned slightly opaque and thena small amount of Compound 1E [DSJ-F-20(15)] was added to inducecrystallization. The resulting slurry was filtered washing with waterand was dried overnight under vacuum giving 5.46 g of Compound 1E[MDM-W-26(8), 65% yield, 99.9 area % by HPLC, 98.6 wt % by ₁H NMR].

F. N-Benzylation was evaluated at temperatures ranging from 25° C. to75° C. to determine the optimal temperature for the reaction and itsthermal tolerance. The reaction rate increased with temperature, but allapproached a common endpoint of 95-96% conversion after 20 hoursregardless of temperature. Analysis by HPLC showed little difference incrude purity, but a notable color change occurred above 45° C. renderingthe reaction solution as light orange. A reaction temperature of 45° C.was considered optimum in terms of reaction rate and diminished colorchange and precipitate shelling.

G. The process was amended by increasing the amount of benzyl chlorideto 1.1-1.15 equivalents and by slightly lowering the reactiontemperature to 45° C. to reduce discoloration. An aqueous workup afterthe N-benzylation was incorporated to remove impurities generated duringthe N-benzylation reaction prior to formation of 1E. Isolation of theproduct was done by direct crystallization from the reaction mixture(isoamylamine) by adding water as an antisolvent. Loss of product to thefiltrate was typically less than 7%. 1E was isolated as a white solid inapproximately 80% yield at very high purity.

EXAMPLE 5 Complete Performance of Scheme 1

A. A 50 g sample of 2-chloronicotinic acid was treated with N-Bocpiperazine (1.2 equiv) in the presence of triethylamine (2 equiv) andTBTU (1.4 equiv). The reaction was carried out in IPAc (300 mL, 6 vol)and at ambient temperature. The reaction was judged complete by HPLCanalysis after 12 hours. After filtration and aqueous workup, the IPAcextract was reduced to 180 g under vacuum (≈1:1 IPAc/product by weight).

To the resulting slurry was added heptanes (≈1:1 IPAc/product byweight). The suspension that resulted was stirred at ambient temperaturefor 16 hours and for another two hours in an ice-water bath. The productwas collected by filtration on a Whatman® #1 filter paper and the filtercake was washed with heptanes (2×25 mL). The product was dried in avacuum oven at ambient temperature to obtain 78.53 g (76% yield) ofcompound 3EPr as a brown solid. HPLC purity was 98.9% (AUC @ 226 nm).

B. 73.53 g of compound 3EPr obtained in paragraph A was subjected to theBoc-deprotection reaction in the presence of 2 equivalents of 5-6 N HClin IPA. The reaction was carried out at 50° C. in IPA (6 vol). Thereaction was judged complete by HPLC analysis after seven hours. Thereaction mixture was cooled to ambient temperature and was filteredthrough a Whatman® #1 filter paper. The filter cake was washed with IPA(2×50 mL) and dried under high vacuum to obtain 56.31 g (95% yield) of3E.HCl as a brown solid. HPLC purity was >99.9%.

C. A sample of 54.0 g of 3E.HCl was then treated with benzyl chloride(1.05 equiv) in the presence of triethylamine (3 equiv). The reactionwas carried out at 50° C. in acetonitrile (6 vol). The reaction wasjudged complete by HPLC analysis after eight hours. The reaction mixturewas cooled to ambient temperature and the solids were filtered on aWhatman® #1 filter paper. The filter cake was washed with acetonitrile(2×25 mL). The solvent was removed under reduced pressure to adjust theweight of the solution to 110 g (≈1:1 acetonitrile/product by weight).

To this was added isoamylamine (220 g) to make 4:1isoamylamine/acetonitrile. The resulting solution was heated to 85° C.and the reaction was judged complete by HPLC analysis after 22 hours.The reaction mixture was cooled to ambient temperature and the solventwas removed under reduced pressure to adjust the weight of the solutionto 150 g. To the resulting mixture was added heptanes (6 vol). Thesuspension was stirred at ambient temperature for 16 hours and thesolids were collected by filtration on a Whatman® #1 filter paper andthe filter cake was washed with heptanes (250 mL×2), followed by water(250 mL×2). The product was dried in a vacuum oven at ambienttemperature to afford 60.66 g (80% yield over two steps) of 1E as alight brown solid. HPLC purity was >99.9% (AUC @ 226 nm).

EXAMPLE 6 Preparation of Phosphate Salt

A. A 22 L, three-neck, round-bottom flask equipped with an additionfunnel, a reflux condenser, a thermocouple, and an overhead stirrer wasplaced in a heating mantle. The flask was charged with ethanol (7.9 L,Pharmco lot #0802062) followed by deionized water (420 mL). Next 1E (700g, 2.1 mol) was charged to the reactor and the resulting mixture washeated to 75° C. A 1 M solution of H₃PO₄ in ethanol (4.5 L, 4.5 mol, 2.1equiv) was charged as a quick stream over a period of 30 min. Theresulting mixture was stirred for 15 min and 1E.H₃PO₄ (0.5 g) was addedas seed for recrystallization. The resulting clear solution was cooledto ambient temperature at a rate of 20° C./h.

The cooled suspension was allowed to stir at ambient temperature for 11h and filtered through a Whatman® #1 filter paper. Ethanol (2.8 L×2) wasused to aid in the transfer and also to wash the filter cake. Theproduct was dried under vacuum to a constant weight at 25° C. to obtain1E.H₃PO₄ as a white solid (751 g, 62% yield). Analysis by HPLC indicateda purity of >99.9% (AUC @ 226 nm) and ¹H NMR was consistent with theassigned structure.

B. Compound 1E (4.9 g, 13.3 mmol) was dissolved in a 5% mixture of waterin ethanol at 75° C. and then 1 M phosphoric acid in ethanol (2.1 equiv)was added. The resulting solution was cooled to ambient temperature at arate of 20° C./h during which time a sticky precipitate formed. Themixture was reheated to dissolve the precipitate, and then the systemwas seeded with 1E phosphate and cooled as described above. Theresulting slurry was stirred overnight at ambient temperature and thenfiltered, washing with ethanol to give 4.9 g of 1E phosphate (79%yield, >99.9 area % by HPLC) as a white solid. The results indicatedthat seeding was essential to establishing the proper crystal form.

C. Four phosphate salt-formation reactions were conducted on a 10 gscale under the following conditions:

MDM-W-126: 1.25 equivalents H₃PO₄, 12 volumes EtOH

MDM-W-128: 1.25 equivalents H₃PO₄, 12 volumes 5% water in EtOH

MDM-W-130: 1.0 equivalent H₃PO₄, 12 volumes EtOH

MDM-W-131: 1.0 equivalent H₃PO₄, 12 volumes 5% water in EtOH

Each reaction was heated to 70° C., seeded with 1E phosphate [0.1 wt %,DAJ-F-40(2)], and cooled to 20° C. at a rate of 20° C./hour. Theresulting thick slurry was stirred overnight, filtered (washing withEtOH), and dried to a constant weight. The results of these reactionsare shown in Table 1. In general, the slurry obtained from reactionsusing 5% water in EtOH was more manageable.

TABLE 1 Phosphate Salt Formation Screen Reaction % Yield Purity (Area %)Potency (wt %)* MDM-W-126 97 >99.9 105 MDM-W-128 95 >99.9 105 MDM-W-13096 >99.9 102 MMD-W-131 94 >99.9 103 *Potency relative to NCSS batchDAJ-F-40(2).

Physical Properties of the Mono-Phosphate Salt

Solubility in water was >36 mg/mL under ambient conditions, and the saltwas crystalline by XRPD analysis.

DSC analysis showed one endothermic event at 179° C. which is consistentwith a melt.

Moisture sorption analysis showed the material was moderatelyhygroscopic, adsorbing 4.4 wt % water at 60% RH and 11.2 wt % at 90% RH.

IC analysis showed a formula (1) to counter-ion ratio of 1:1.6 to 1:2.3in different batches of the salt.

1. A method to convert a compound of formula (1):

to a phosphate salt by reacting said compound with phosphoric acidwherein the reaction occurs in a solvent mixture consisting of ethanoland water.